The synthesis of polypeptides by conventional solid-phase methods, amino acid by amino acid, is limited by low yields when the polypeptides synthesized are of large size. For overcoming this limitation, assembly of two polypeptides by chemical ligation, in order to produce a longer polypeptide, is known.
In general, it is desirable for the bond between the polypeptides assembled by ligation to be native, i.e. to correspond to the natural structure of the polypeptides.
Currently the main method for native ligation is that of Kent and Dawson, which is described for example in documents WO 96/34878 and WO 98/28434. This method is based on a chemoselective reaction between a (C-terminal) thioester peptide and a cysteinyl peptide. The main drawback of this method is that manufacture of the thioester peptides requires complex chemical processes.
An alternative method is so-called Staudinger ligation, described in documents WO 01/68565 and WO 01/87920. This comprises reaction of a phosphinothioester with an azide and hydrolysis of the combined reagents to form an amide bond. This method is difficult to apply on an industrial scale.
A third method, described in document WO 2007/037812, is based on reaction of an α-keto acid with an amine in a reaction of decarboxylative condensation. However, the keto acids are molecules that are difficult to manufacture and to incorporate in peptides. Moreover, this third method is difficult to apply in peptide synthesis laboratories that are not equipped with means for carrying out complex organic syntheses.
There is therefore a real need to develop a new method for native ligation of polypeptides, which is both effective and simpler to implement, including on an industrial scale.